Methods for Purifying Biological Cells

ABSTRACT

This invention relates to devices and methods for purifying biological cells. For example, viable tumor, stem, immune and sperm cells can be purified from a complex biological sample using a pipette tip column. Methods of the invention can aid research, diagnosis and treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 14/181,656, filed Feb. 15, 2014, which claims the benefit ofU.S. Provisional Application No. 61/765,541, filed Feb. 15, 2013, whichis incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to devices and methods for purifying biologicalcells. For example, viable tumor, stem, immune and sperm cells can bepurified from a complex biological sample using columns, such as pipettetip columns.

SUMMARY OF THE INVENTION

In the present invention, cells are purified from a biological sampleusing a column. The sample is passed through the column and cells arecaptured on the medium within the column. Following capture, the columnis washed to remove material that is not specifically bound to thecolumn medium. Cells are recovered by passing an eluent through thecolumn.

BACKGROUND OF THE INVENTION

The primary technology for capturing cells is magnetic beads. In thistechnology, a suspension of beads is used to treat a sample containingcells. The magnetic beads contain a tag or chemical entity that isselective for cells or for a certain cell type within the sample. Afterthe cells become associated with the magnetic beads, a magnet is used tocollect the magnetic beads and captured cells. The magnetic beads may bere-suspended several times with wash solutions to clean the cells.Finally, a solution may be used to release the cells from the beads. Amagnetic is used to separate the magnetic beads from the cells.

The magnetic-activated cell sorting (MACS) method available fromMiltenyl Biotec allows cells to be separated by incubating with magneticnanoparticles coated with antibodies against a particular surfaceantigen. Cells expressing this surface antigen attach to the magneticnanoparticles. Afterwards the cell solution is transferred on a columnplaced in a strong magnetic field. In this step, the cells attached tothe nanoparticles (expressing the antigen) stay on the column, whileother cells (not expressing the antigen) flow through.

With the MACS method, the cells can be separated either positively ornegatively with respect to particular antigens. With positive selection,cells expressing the antigen(s) of interest, which attached to themagnetic column, are washed out to a separate vessel, after removing thecolumn from the magnetic field. This method is useful for isolation of aparticular cell type, for instance CD4 lymphocytes. In negativeselection, the antibody used is directed against surface antigen(s)known to be present on cells that are not of interest. Afteradministration of the cells/magnetic nanoparticles solution onto thecolumn the cells expressing these antigens bind to the column andfraction that goes through is collected, as it contains almost no cellswith undesired antigens.

Rare circulating tumor cells (CTCs) present in the bloodstream ofpatients with cancer provide a potentially accessible source fordetection, characterization, and monitoring of nonhematological cancers.A microfluidic device, the Harvard CTC-Chip, has been used to capturingthese epithelial cell adhesion molecule (EpCAM)-expressing cells usingantibody-coated microposts. In a first generation device called 78,000antibody-functionalized microposts were used to separate cells. Anothermicrofluidic mixing device called the herringbone-chip is made up ofparallel slanted channels (Li et al, Lab Chip, 13, 602).

In one technology, cells are collected on a flow through column at veryslow flow rate and very low volumes making them difficult to use. Thechip column does not contain a frit because the frit would preventpassage of the cells or trap, damage or kills the cells. But instead ofa column frit a thin passage channel was used at the base of the chip tolet the liquid flow through the column (Kralj et al. Lab Chip, 2012, 12,4972-4975).

The EpCAM-based technique is very low throughput. A suspension of wholeblood was pumped from 3 mL syringes at 0.2 mL/h for 1 h through themicrofluidic packed bed to allow immobilization of the cancer cells. Atthis rate 5 hours is need to process 1 mL of blood. This may be animprovement over the Harvard CTC-chip, but still does not solve somefundamental issues with EpCAM based capture. The throughput isimpractical and it too slow. Multiple channels can be used, but thatwill dramatically increase the imaging and staining area, making itdifficult to perform high resolution cytomorphological analysis

These magnetic bead methods and micro or chip based columns are slow anddo not produce pure cell populations. There exists a need for a columntechnology that captures cells at high concentrations and then recoversthe cells at high purity.

BRIEF DESCRIPTION OF THE INVENTION

In this work, we describe an invention for the separation andpurification of cells, including viable cells and cancer cells. Abiological sample containing cells is passed through a column. In someembodiments, the column is a pipette tip column. In some embodiments,the flow of sample through the column is bidirectional. In someembodiments the flow rate is quite high so that the cell purificationcan be performed in less than 1 hour. In certain embodiments, cells canbe purified from a sample in less than 30 minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B. Stylistic depiction of flow path, nooks and traps in acolumn. FIG. 1A depicts an aspiration step. FIG. 1B depicts an expulsionstep.

FIG. 2. Elution of cells from a column shown in FIG. 1B.

FIGS. 3A-B. Depiction of the column and method of the invention. FIG. 3Adepicts and aspiration step and FIG. 3B depicts and expulsion.

DETAILED DESCRIPTION OF THE INVENTION

It is quite remarkable that intact cells and even viable cells can bepurified using the columns and methods of the invention. Cells aredefined herein as membrane-bound structures that occur as functionalindependent units of life (such as in unicellular organisms, e.g.bacteria, protozoa, etc.), or as structural or fundamental units in abiological tissue specialized to perform a particular function inmulticellular organisms (e.g. plants and animals).

Cells are quite fragile and can rupture easily from a variety ofphysical conditions such as encountering an object, shearing force,turbulence or incorrect solute concentration. Mechanical cell lysis canbe induced by a collision of the cells with micro beads. In fact, thisis a common method for lysing cells. However, even a little damage, evenone breach of the cell membrane is enough to cause catastrophic damageto a cell. Viable cells can die simply from incorrect storage,processing, transport, exposure to incorrect temperature (heat or cold),pH, medium, vessel, etc.

It is surprising that cells can remain intact even after subjecting themto the methods of the invention. Specifically, cells purified via theinstant invention are subjected to a repeated back and forth flow,battering motion through a fritted column containing a bed of medium.Cells are flowed through a fritted column containing a bed of medium.Most commercially-available methods for cell isolation involve the useof magnetic beads. To the inventors' knowledge, cells have never beenisolated from fritted columns using the flow rates disclosed herein orusing back and forth flow.

As described above, the columns of the invention contain a bed of mediumonto which the cells are captured. The bed can be comprised of beads orparticles held in the column by at least one frit. In certainembodiments, the bed is retained in the column with two frits; one belowthe bed and one above the bed. It is quite surprising that cells canpass through the frit(s) and the bed of medium and maintain theirintegrity and in some cases, their viability.

Consider the physical environment of a liquid sample comprised of cellspassing through the frit and bed of medium within a column. The channelsthrough which a cell might flow are not open or linear. Instead, theflow path would consist of a variety of interwoven channels, each withvarying and perhaps restrictive diameters, and many possible dead endsmarked by repeated turns, bends, winding and twisting. This tortuouspath environment is advantageous for the capture of small moleculeanalytes because the fluid (containing the analyte) gets extensiveexposure to the column matrix. However, a cell travelling or flowingthrough this environment could easily be trapped. Of course, physicallytrapping cells within the column matrix is an undesirable outcome quitedistinct from targeted cell capture strategies such as affinity binding.Cells that become physically trapped cannot be recovered with an eluentor desorption solvent. Furthermore, if cells are trapped eventemporarily, they could readily rupture or die.

FIGS. 1A and 1B illustrate the surprising nature of the invention. It isa stylistic depiction of the many potential hazards and pitfalls thatcould be encountered by cells travelling through a column usingbidirectional flow. FIG. 1A depicts an aspiration step in which the flowdirection 10 is upward. The matrix of the material (e.g., a polymer) isdepicted by closed squares 16. Cells cannot penetrate matrix 16. Theflow path through a column bed contains many potential nooks and trapsfor cells. A clear unrestricted flow path 12 enters and exits the bed.Some cells (e.g., 14) may be captured by the column in flow path 12. Asthe flow proceeds, many or most of the cells 18 enter dead end flowpaths 20 to trap the cells 22 in dead end or restricted passages 24.There are also nooks (e.g., 26) just off flow path 12 that may trap cell28.

FIG. 1B depicts the fate of cells resulting from back and forth flowthrough the column. The flow direction 30 is in a downward direction,reversed from upward direction 10 shown in FIG. 1A. Although increasedresidence time may allow a greater number of cells 32 to be captured,this reversal of the flow direction 34 can also exacerbate the undesiredtrapping of many cells 36. It should be noted that cell 28 remainstrapped in nook 26.

FIG. 2 depicts the elution of cells from a column. The recovery of cellsfrom a column is attempted with a downward flow direction 38. Most ofthe cells 40 remain irreversibly trapped. A few cells 42 may berecovered but may or may not be intact. In addition to the risk of celltrapping, a person of skill in the art would expect the columnenvironment or materials to be inhospitable to cells. It is desirable torecover intact and even viable cells. Intact cells are defined herein ascells having no holes or ruptures in their membrane. The columnmaterials or surfaces, such as the frit or column walls might beincompatible with the cell integrity or viability. Protrusions presentin the column wall, bed or frit could easily damage or rupture cells.

FIGS. 3A and 3B depict a column and method of the invention. Cells in aliquid sample are passed through the column using back and forth flow.In these embodiments, the upper end of the column is operatively engagedwith pump 40 and sample 46 containing cells 44 is aspirated and expelledthrough the lower end of the column. During the aspiration step, thesample travels in direction 52, upwards in through lower frit 56 intothe bed of beads 48 and then continues through upper frit 50 (FIG. 3A).During expulsion, the sample 46 travels back downward in direction 54,back through upper frit 50, into the bed of medium, through lower frit56 and exits the bottom of the column (FIG. 3B). These aspirations andexpulsions can be repeated multiple times, the desired result being thatintact cells are captured by the medium.

Although column flow is harmful to cells, intuitively, it seems that theflow paths resulting from back and forth flow would be even moreperilous for cells than unidirectional flow, especially when the goal isrecovery of intact cells or viable cells. Cells would pass through thefrit(s) multiple times from both directions, increasing the probabilityof cell damage or death.

Even when slow rates are used, cells can travel through the column atrelatively high linear velocities. A high linear velocity would beexpected to exacerbate the potential problems listed above. For example,a cell could become lodged in a dead end with greater force, making itmore difficult to free the cell. While a cell travelling at a relativelyslow velocity might slide or sidle around an obstacle, a cell travellingat a high velocity might be ruptured.

The starting sample contains viable cells and can be from any biologicalsource. For example, cells can be captured from biological fluids suchas blood or semen or tissues such as brain or tumor tissue. Theinvention is not limited to particular tissue type or cell type; cellscaptured by the methods of the invention can be eukaryotic orprokaryotic cells. In certain embodiments, the method can be used forthe analysis of cells from crime scene samples.

In some embodiments, the columns used for the methods of the inventionare pipette tip columns. Pipette tip columns are defined herein ascolumns capable of operative engagement with a pipette or liquid handingrobot. In some embodiments, the columns can be integrated into amulti-well plate. In other embodiments, the columns are adapted toengage a syringe or syringe pump.

The method can be performed in an automated or semi-automated fashion.The term “semi-automated” is defined as a process by which two or moresamples, columns or tubes are processed simultaneously or by which thesample process is at least partially performed by a timed computer orprocessor controlled program. The term “automated” is defined as aprocess by which sample processing is performed by a robotic systemcontrolled by a timed computer program.

The columns of the invention are comprised of a medium on which thecells are captured. The medium can be beads or particles. In certainembodiments, the column medium can be a monolith, a filter or acombination of materials. In some embodiments, the bead size is quitelarge, on the order of 100-900 microns or in some cases even up to adiameter of 3 mm. In other embodiments, the bead size is that used inconventional columns, on the order of 45-150 microns. The averageparticle diameters of beads of the invention can be in the range ofabout 20 μm to several millimeters, e.g., diameters in ranges havinglower limits of 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm,100 μm, 150 μm, 200 μm, 300 μm, or 500 μm, and upper limits of 20 μm, 30μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm,300 μm, 500 μm, 750 μm, 1 mm, 2 mm, or 3 mm.

Various mechanisms can be used for cell capture on the medium.Non-limiting examples include a functional group that has affinity forthe cells, use of a tagged antibody, ion exchange, a tagged aptamer andan antibody loaded resin (Pro A, G etc.), covalently bonded linkers(alkyl thio, etc.) and hydrogen bonded linkers.

The medium may be held in the column with at least one frit positionedbelow the bed of medium. This lower frit may lie at the lower end of thecolumn or it may be positioned some distance above the lower end. Incertain embodiments, a second frit is positioned above the bed. The bedcan be packed between two frits using a light force packing method. Thegoal of light force packing is not to compact the bed or introducerestrictive flow channels. In alternative embodiments, the column lacksa top frit. In still other embodiments, there is a gap between the bedof medium and the top frit. This gap is referred to as an air gap.

The column frits should have a pore size small enough to contain themedium but large enough for cells to pass through. Frits of theinvention preferably have pore openings or mesh openings of a size inthe range of about 5-500 μm. In preferred embodiments, the frits can bequite thin to lessen the probability that cells will become trappedwithin the frit. It is important that the frit does not provide dead-endor restricted-end flow paths that could potentially trap or damagecells. In some embodiments, a screen or fabric frit is utilized.

In some embodiments, the sample is aspirated and expelled through thelower end of the column. This method is referred to as back and forth orbidirectional flow. In these embodiments, a pump, such as a liquidhandling robot is operatively engaged with the upper end of the columnand the sample is aspirated and expelled through the lower end of thecolumn. In other embodiments, unidirectional flow is used. In theseembodiments, the sample is added to the upper end of the column andflows in a downward direction through the column and out the lower end.The sample can be passed through the column with the use of a pump, avacuum or even gravity.

A relatively high solvent flow rate having high linear velocity can beused with the methods of the invention. Columns of the invention canaccommodate a variety of flow rates, and the invention provides methodsemploying a wide range of flow rates, oftentimes varying at differentsteps of the method. In various embodiments, the flow rate of liquidpassing through the media bed falls within a range having a lower limitof 0.01 mL/min, 0.05 mL/min, 0.1 mL/min, 0.5 mL/min, 1 mL/min, 2 mL/min,or 4 mL/min and upper limit of 0.1 mL/min, 0.5 mL/min, 1 mL/min, 2mL/min, 4 mL/min, 6 mL/min, 10 mL/min or greater. For example, someembodiments of the invention involve passing a liquid though a packedbed of media having a volume of less than 100 μL at a flow rate ofbetween about 0.1 and about 4 mL/min, or between about 0.5 and 2 mL/min,e.g., a small packed bed of extraction media as described elsewhereherein. In another example, other embodiments of the invention involvepassing a liquid though a packed bed of media having a volume of lessthan 25 μL at a flow rate of between about 0.1 and about 4 mL/min, orbetween about 0.5 and 2 mL/min. Column with larger bed volumes e.g. 200μL to 10 mL and 1 mL to 50 mL may use even faster flow rates.

Columns used in the invention contain material capable of capturingcells. Cells are large and bulky. As cells flow through the column,their orientation may not be ideal making it difficult to capture themwith affinity groups. In certain embodiments, the columns are comprisedof a packed bed of medium. In other embodiments, the columns can containa bed that is not tightly packed or fluidized. The medium can becomprised of beads or particles. When a sample containing viable cellsis passed through the column, the cells are captured by the materialwithin the column. In some embodiments, the column is operated in a coldroom while in other embodiments, the column can be operated at roomtemperature or at a temperature greater than room temperature.

After the capture step, the columns are washed with buffer or water toremove any material that is not specifically bound to the column medium.The wash liquid can be passed through the column by any means or ratedescribed above for the sample. The wash step may be repeated once toseveral times.

Following the column wash, the cells can be eluted from the column bypassing an eluent through the column. The eluent can be passed throughthe column by any means described above for the sample. The elution stepmay be repeated once to several times. In certain embodiments, theeluent is incubated on the column for a period of time to increase theefficiency of cell elution. After the purified cells are eluted from thecolumn, they can be analyzed by any means desired.

In some embodiments, the cells are not eluted from the column. Instead,they can be lysed on column or the column bed material with cells boundcan be released from the column and subjected to further analysis suchas a polymerase chain reaction.

Non-limiting examples of how the columns can be used include thefollowing.

1) capture and release of cells2) depletion of cells from a complex cellular mixture and retention ofremaining cells3) capture labeled cells, then release and count4) Capture cells, lyse cells on column or post column. Collect DNA orRNA by DNA prep or RNA prep, or collect proteins or other cellularcomponents and analyze5) Purify sperm from a crime scene away from other cells and perform DNAanalysis to identify the source of the sperm6) After cell capture, the column is washed and cells can optionally bereacted with a dye to label the captured cells. The resin may be removedfrom the column and plated or spread on a surface. The resin beadscontaining attached cells may be sorted and counted or analyzed by anymeans.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover and variations,uses, or adaptations of the invention that follow, in general, theprinciples of the invention, including such departures from the presentdisclosure as come within known or customary practice within the art towhich the invention pertains and as may be applied to the essentialfeatures hereinbefore set forth. Moreover, the fact that certain aspectsof the invention are pointed out as preferred embodiments is notintended to in any way limit the invention to such preferredembodiments.

EXAMPLES Example 1 Sperm is Captured, Separated from Cells and DNAAnalysis is Performed

In forensics, it is often desired to obtain DNA profiles from oldstains, body fluid samples and other possible samples. The primary goalis to preferentially separate sperm from vaginal cells and othermaterials, a necessity for DNA analysis in rape cases, for example.

DNA aptamers which are short strands of DNA were developed by SomaLogic(Denver, Colo.) to bind sperm heads, and used to both identify andimmobilize the sperm heads for purification and later DNA analysis.These aptamers are used in a column bed system of the invention withBiotin and Streptavidin linkers to selectively capture sperm cells. Theaptamer sequences bind preferentially to both the outer protein membraneand the stripped perinuclear calyx of sperm cells in the presence ofnon-sperm epithelial cells.

Sperm cells (research vials, prepared by density gradient centrifugationand subsequent washing) are purchased from California Cryobank. Washedsperm cells are prepared using three washes and suspension in a buffersupplemented with Triton X100 detergent and NaCl to final concentrationsof 1% v/v and 600 mM. HeLa cells to simulate non-sperm epithelial cellsare added and the mixtures are incubated for ten minutes.

Cotton swabs are used to simulate capturing the sperm sample. The sampleis removed from the cotton swab with a buffer. Aptamers with biotinlinkers are added to the solution and incubated. After washing of thesample the mixture is passed through a streptavidin packed bed column ofthe invention. The sperm is captured and subsequently washed by passingwash buffer through the column.

The sperm is eluted from the column by passing a buffer through thecolumn breaking up the aptamer/sperm column. Eluted aptamer DNA arepurified and then amplified for DNA analysis.

Example 2 Antibody Purification of Sperm

This example uses antibodies rather than aptamers to capture sperm cellsin the presence of other cells. A cocktail of antibodies specific tosperm cell surface antigens are anchored to Protein A affinity beadspacked into a column of the invention. The specificity ofantibody-antigen binding selectively captures sperm cells from samplesthat are comprised of a mixture of sperm cells, white blood cells,epithelial cells, cell lysates, etc. Alternatively, the antibodies areadded to the sample mixture first and then captured by the column. Afterwashing with a neutral buffer, the sperm cells are eluted with low pH orhigh pH buffers and the DNA is analyzed.

The antibodies may be tagged with His tags for example. In this case,IMAC beads may be packed into columns of the invention to capture theantibodies which are used in turn, to capture the sperm. In this case,the antibody sperm combination may be eluted, the cell lysed and the DNAanalyzed. Other tags may be used such as FLAG-ANTIFLAG, etc.

Peptide tags are used for capture. These include AviTag, a peptideallowing biotinylation by the enzyme BirA so the protein can be isolatedby streptavidin, Calmodulin-tag, a peptide bound by the proteincalmodulin, FLAG-tag, a peptide recognized by an antibody, HA-tag, apeptide recognized by an antibody, Myc-tag, a short peptide recognizedby an antibody, SBP-tag, a peptide which binds to streptavidin, Softag1, for mammalian expression, Softag 3, for prokaryotic expression, V5tag, a peptide recognized by an antibody, and Xpress tag.

Covalent tags include Isopeptag which binds covalently to pilin-Cprotein and SpyTag which binds covalently to SpyCatcher protein.

Protein tags include BCCP (Biotin Carboxyl Carrier Protein), a proteindomain recognized by streptavidin, glutathione-S-transferase-tag, aprotein which binds to immobilized glutathione, green fluorescentprotein-tag, a protein which is spontaneously fluorescent and can bebound by nanobodies, maltose binding protein-tag, a protein which bindsto amylose agarose, Nus-tag, Strep-tag, a peptide which binds tostreptavidin or the modified streptavidin called Strep-Tactin andThioredoxin-tag.

Example 3 Circulating Tumor Cells

A cancerous tumor sheds small numbers of tumorous cells into itsimmediate vasculature. These cells then make their way into thecirculatory system, and are thus called circulating tumor cells (CTCs).CTC information is used cancer prognosis, therapy monitoring andmetastasis research.

Circulating tumor cells (CTCs) are important targets for study tounderstand, diagnose, and treat cancers. However, CTCs are found inblood at extremely low concentrations which makes isolation, enrichmentand characterization challenging. A typical concentration in a humancancer patient is approximately 1-100 CTCs per mL of blood.

CTC purification with the columns of the invention capture many or mostof the CTCs in the blood sample (high capture efficiency) and areselective with very few other cells accidently isolated. The samples areprocessed with sufficient speed and without battering of the cells sothat cells remain viable in many cases.

The columns of the invention operate by coating the column media with anantibody (anti-EpCAM) and then bonding the antibody to the epithelialadhesion molecules (EpCAM) of CTCs. After capture of anti-EpCAM labeledCTCs from a blood sample, CTC identification and enumeration areachieved using immunostaining.

During one experiment 2 to 80 spiked breast cancer cells are isolatedfrom 1 mL of mice blood sample with 90% capture efficiency. A 200 μL bedcolumn with a 1 mL pipette tip body is used for one experiment. Whileblood is processed through the column with bidirectional flow for 5cycles at 200 uL/min flow rate. The column is washed with buffer andthen the cells are eluted with 500 mM citric acid.

Example 4 Capture of Cells from Blood

The purification and analysis processes used in example 3 are used forother cells from blood including white blood cells, stem cells, T cells,B cells and other cells.

Example 5 Capture of Cells from Blood

The purification and analysis processes used in examples 3 and 4 areused except the pumping methods for flowing the fluids through thecolumn are changed as follows.

The pumping method is bidirectional, unidirectional, gravity flow andgravity flow plus vacuum and/or pressure.

In addition, the method is performed with two different columnconfigurations. In one configuration, there is an air gap above thecolumn bed, while in the other configuration, there is no air gap abovethe column bed.

Example 6 Capture of Cells from Blood

The purification and analysis processes used in examples 3, 4 and 5 areused except the column has a bed volume of 1 mL inside a 10 mL pipettebody. The flow rates are approximately 10 times faster with this columnso samples sizes approximately 10 times greater are processed inapproximately the same time.

In this example the cells are released from the column by enzymatic andchemical cleavage of the linker. The cells are collected and counted.

Examples 7 Capture of Cells from Tissue

For tissue samples composed of different types of cells, heterogeneouscell populations will be present. To obtain as much information aspossible about an individual cell type, biologists have developed waysof dissociating cells from tissues and separating the various types. Amild procedure is used to collect whole, intact cells. Homogenized cellsare kept at low temperatures to prevent autolysis and kept in anisotonic solution to prevent osmotic damage.

The first step in isolating cells of a uniform type from a tissue thatcontains a mixture of cell types is to disrupt the extracellular matrixthat holds the cells together. For example, viable dissociated cells areobtained from fetal or neonatal tissues. The tissue sample is treatedwith proteolytic enzymes (such as trypsin and collagenase) to digestproteins in the extracellular matrix and with agents (such asethylenediaminetetraacetic acid, or EDTA) that bind, or chelate, theCa²⁺ on which cell-cell adhesion depends. The tissue can then be teasedapart into single living cells by gentle agitation to make a cellsuspension.

Columns of the inventions are loaded with antibodies that have anaffinity for fetal cells. The suspension is passed with bidirectionalflow through the column to capture the cells. After washing, the cellsare released with by treatment with trypsin to digest the antibodies.The cells may be visually tagged by using an antibody coupled to afluorescent dye to label specific cells.

Given appropriate surroundings, most plant and animal cells can live,multiply, and even express differentiated properties in a tissue-culturedish. The cells can be watched continuously under the microscope oranalyzed biochemically, and the effects of adding or removing specificmolecules, such as hormones or growth factors, can be explored. Inaddition, by mixing two cell types, the interactions between one celltype and another can be studied. Experiments performed on cultured cellsare sometimes said to be carried out in vitro (literally, “in glass”) tocontrast them with experiments using intact organisms, which are said tobe carried out in vivo (literally, “in the living organism”). Theseterms can be confusing, however, because they are often used in a verydifferent sense by biochemists. In the biochemistry lab, in vitro refersto reactions carried out in a test tube in the absence of living cells,whereas in vivo refers to any reaction taking place inside a living cell(even cells that are growing in culture).

Example 8 Capture of Bacterial Cells

An E. coli culture is grown at 37° C. The E. coli strain is engineeredusing recombinant DNA techniques so that surface proteins on the cellcontain histidine tags. A spike of Salmonella is added to the sample sothat the sample contains 10% Salmonella cells, 90% E. coli cells, mediaand other materials.

A 1 mL bed size column containing Ni form IMAC affinity media is used totreat or process a 3 mL sample with unidirectional single pass flowunder gravity. Some air pressure is used to push the last remainingsolution through the column. The E. coli cells are removed from themixture and are captured on the column while the Salmonella cells remainin the sample.

Example 9 Capture of Cells from Culture

Most plant and animal cells can live, multiply, and even expressdifferentiated properties in a tissue-culture dish. The cells can bewatched continuously under the microscope or analyzed biochemically, andthe effects of adding or removing specific molecules, such as hormonesor growth factors can be explored. In addition, by mixing two celltypes, the interactions between one cell type and another can bestudied. After growing the cells, the specific cells are capturedaccording to processes similar to those described in examples 7 and 8.

After capture, the column is washed and optionally reacted with a dye tolabel the captured cells. The resin may be removed from the column andplated or spread on a surface. The resin beads containing attached cellsmay be sorted and counted or analyzed by any means.

Example 10 Companion Diagnostic to Antibody or FAB Based Drug

Often it is unknown whether a particular antibody or FAB drug will beeffective against a particular cancer case. The treatment process can betrial and error, trying one drug and then if it is not effective, tryingthe next drug and so on. Columns of the invention may be used todetermine the potential effectiveness of a series of drugs. Tagged drugantibodies and FABs are prepared. A series of columns of the inventionare prepared each with a single antibody bound through the tag to themedia of the column. In this way, each available drug is represented bya column. Then a blood sample from a cancer patient is treated by theseries of columns in an attempt to capture circulating tumor cells. Thecolumns are washed and the cells, if present, are recovered and analyzedby fluorescence, DNA, microscopy or any suitable analytical technology.Specific drugs that may be effective against the cancer are capturedcontaining drug based affinity media. Then a treatment program isdesigned using the antibody/FAB drugs that have the highest affinity forthe tumor.

What is claimed is:
 1. A method for purifying viable cells, comprisedof: a) providing at least one column, wherein the column is comprised ofa packed bed of medium, wherein the packed bed of medium has a bedvolume, wherein the packed bed of medium is retained between two frits,a lower frit and an upper frit, wherein the packed bed of medium iscomprised of particles, wherein the particles are comprised of anaffinity group, wherein the affinity group is an antibody; b) providinga biological sample comprised of viable cancer cells, wherein the viablecancer cells are not labeled with antibodies, wherein the biologicalsample has a sample volume, wherein the sample volume is larger than thebed volume; c) aspirating the biological sample in an upward directionthrough column flow, wherein the sample is aspirated through the lowerfrit, into the packed bed of medium and then through the upper frit; d)expelling the biological sample by reversing the flow direction, whereinthe biological sample is expelled in a downward direction through theupper frit, into the packed bed of medium and then through the lowerfrit; e) repeating steps (c) and (d) multiple times, wherein thebiological sample is not incubated on the column, whereby a portion ofthe viable cancer cells are captured by the antibody affinity groups onthe particles in the packed bed of medium; f) aspirating a wash solutionthrough the column using bidirectional flow, wherein the wash solutionis aspirated through the lower frit, into the packed bed of medium andthen through the upper frit; g) expelling the wash solution through theupper frit, into the packed bed of medium and then through the lowerfrit; and h) eluting the cancer cells by passing an eluent through thecolumn, wherein the eluted cancer cells are intact and viable, whereinthe eluted cancer cells maintain their integrity, wherein the method isat least partially performed by a timed computer-controlled program, andwherein the entire method is performed in less than one hour.
 2. Themethod of claim 1, wherein the column is a pipette tip.
 3. The method ofclaim 1, wherein the particles are comprised of tagged antibodies. 4.The method of claim 3, where in the tag is a drug.
 5. The method ofclaim 1, wherein the antibodies are covalently attached to the particleswith the linker.
 6. The method of claim 1, wherein the sample containscirculating tumor cells and the antibodies are anti-EpCam.
 7. The methodof claim 1, wherein between steps (g) and (h), the captured cells arelabeled with a dye on the column.
 8. A method for purifying viablecells, comprised of: a) providing at least one column, wherein thecolumn is comprised of a packed bed of medium, wherein the packed bed ofmedium has a bed volume, wherein the packed bed of medium is retainedbetween two frits, a lower frit and an upper frit, wherein the packedbed of medium is comprised of particles, wherein the particles arecomprised of an affinity group, wherein the affinity group is anantibody; b) providing a biological sample comprised of viable bloodcells, wherein the viable blood cells are not labeled with antibodies,wherein the biological sample has a sample volume, wherein the samplevolume is larger than the bed volume; c) aspirating the biologicalsample in an upward direction through column flow, wherein the sample isaspirated through the lower frit, into the packed bed of medium and thenthrough the upper frit; d) expelling the biological sample by reversingthe flow direction, wherein the biological sample is expelled in adownward direction through the upper frit, into the packed bed of mediumand then through the lower frit; e) repeating steps (c) and (d) multipletimes, wherein the biological sample is not incubated on the column,whereby a portion of the viable blood cells are captured by the antibodyaffinity groups on the particles in the packed bed of medium; f)aspirating a wash solution through the column using bidirectional flow,wherein the wash solution is aspirated through the lower frit, into thepacked bed of medium and then through the upper frit; g) expelling thewash solution through the upper frit, into the packed bed of medium andthen through the lower frit; and h) eluting the blood cells by passingan eluent through the column, wherein the eluted blood cells are intactand viable, wherein the eluted blood cells maintain their integrity,wherein the method is at least partially performed by a timedcomputer-controlled program, and wherein the entire method is performedin less than one hour.
 9. The method of claim 8, wherein the column is apipette tip.
 10. The method of claim 8, wherein the captured and elutedblood cells are T cells.
 11. The method of claim 8, wherein theantibodies are tagged with a drug.
 12. The method of claim 8, whereinbetween steps (g) and (h), the captured cells are labeled with a dye onthe column.
 13. The method of claim 8, wherein the particles arecomprised of protein A or protein G.
 14. The method of claim 8, whereinthe antibodies are covalently attached to the particles with the linker.15. A method for purifying viable cells, comprised of: a) providing atleast one column, wherein the column is comprised of a packed bed ofmedium, wherein the packed bed of medium has a bed volume, wherein thepacked bed of medium is retained between two frits, a lower frit and anupper frit, wherein the packed bed of medium is comprised of particles,wherein the particles are comprised of an affinity group, wherein theaffinity group is an antibody; b) providing a biological sample, whereinthe sample is comprised of biological fluids or tissues, wherein thebiological fluids or tissues are comprised of viable cells, wherein theviable cells are not labeled with antibodies, wherein the biologicalsample has a sample volume, wherein the sample volume is larger than thebed volume; c) aspirating the biological sample in an upward directionthrough the column, wherein the sample is aspirated through the lowerfrit, into the packed bed of medium and then through the upper frit; d)expelling the biological sample by reversing the flow direction, whereinthe biological sample is expelled in a downward direction through theupper frit, into the packed bed of medium and then through the lowerfrit; e) repeating steps (c) and (d) multiple times, wherein thebiological sample is not incubated on the column, whereby a portion ofthe viable cells are captured by the antibody affinity groups on theparticles in the packed bed of medium; f) aspirating a wash solutionthrough the column using bidirectional flow, wherein the wash solutionis aspirated through the lower frit, into the packed bed of medium andthen through the upper frit; g) expelling the wash solution through theupper frit, into the packed bed of medium and then through the lowerfrit; and h) eluting the cells by passing an eluent through the column,wherein the eluted cells are intact and viable, wherein the eluted cellsmaintain their integrity, wherein the method is at least partiallyperformed by a timed computer-controlled program, and wherein the entiremethod is performed in less than 30 minutes.
 16. The method of claim 15,wherein the column is a pipette tip.
 17. The method of claim 15, whereinthe captured and eluted cells are stem cells.
 18. The method of claim15, wherein between steps (g) and (h), the captured cells are labeledwith a dye on the column.
 19. The method of claim 15, wherein theantibodies are covalently attached to the particles with the linker. 20.The method of claim 15, wherein the antibodies are tagged with a drug.